ABSTRACT Differentiation therapy is a non-conventional therapeutic modality aimed at re-activating endogenous differentiation programs in cancer cells with subsequent tumor cellular maturation and concurrent loss of the tumor phenotype. The ?curative? power of such therapy has been documented in acute promyelocytic leukemia (APML), but despite multiple attempts to harness the power of differentiation therapy and its popularity as an attractive theoretical option, such therapy has not emerged. Among the reasons cited are- 1) incomplete understanding of the normal stemness-differentiation pathways, and 2) our theoretical inability to pinpoint such a fundamental, actionable and effective target to drive a complex and nebulous process of cancer-to-normal tissue transitioning. Using publicly available transcriptomic datasets from adult and pediatric patients with sporadic and hereditary CRCs and those afflicted with polyposis syndromes and a set of unbiased novel computational approaches (Boolean analysis and Boolean Networks) an unexpected and novel target was identified. These computational approaches, which are designed to identify invariant genes that drive differentiation program in the colon crypts predicted that agonists of the target can trigger differentiation and halt the initiation and progression, and even induce regression of colorectal adenomas and cancers (CRCs), despite disease heterogeneity. Expression pharmacology studies using a companion biomarker in FFPE human tissues confirmed that the pro-differentiation pathway orchestrated by this target is silenced during CRC initiation and progression. Using a potent and highly specific drug that was previously developed for another indication and found to be safe in Phase I trials on healthy human adults, it was confirmed that activation of the target is necessary and sufficient for activation of a pro-differentiation signaling program and in inducing crypt-budding in colon-derived organoids. This proposal seeks to validate the repurposing of a potent and specific drug for activating a novel pro-differentiation target, the first of its kind, in the treatment of colorectal polyposis and cancers. Our specific Aims during the 3-y UG3 phase are all geared towards target validation: obtaining proof- of-mechanism in healthy murine and human colon-derived organoids (Aim 1); preclinical proof-of-principle studies using murine genetic models of CRCs (Aim 2); and expression pharmacology and proof-of-concept Phase ?0? trials in patient-derived organoids (pediatric and adults; Aim 3). Successful demonstration of efficacy in UG3 phase will trigger the UH3-phase (Clinical trial planning; Aim 4). Although the focus here is on pediatric and adult polyposis syndromes and CRCs, network analysis revealed the possibility that the proposed therapeutic/indication pairing may transcend other types of cancers. Much like immunotherapy acts by reinvigorating a physiologic response, the pro-differentiation therapy proposed here promises to reinvigorate yet another physiologic program; it fulfils a much-needed weapon in our anti-cancer armamentarium. Their combined synergy when used as adjuvants may elevate response to ?cure?.